In order to learn more about how membranous secretory vesicles are formed and are induced to discharge neurotransmitters from the synapse, this application proposes to quick-freeze several different sorts of neuromuscular junctions at various intervals after stimulating them electrically. The particular technique of quick-freezing with ultrapure copper cooled to liquid helium temperature developed in this investigator's laboratory over the past few years is designed to prepare the frozen tissues for electron microscopy by modifications of the standard methods of freeze-etching and freeze-substitution. Synapses will be frozen at millisecond intervals after stimulation in order to elucidate how calcium triggers the exocytosis of synaptic vesicles that underlies neurosecretion, and in order to reveal exactly what molecular changes occur during this sort of membrane fusion event. Synapses will also be frozen in the first few seconds after stimulation, in the presence of extracellular tracers such as ferritin and horseradish peroxidase, in order to determine the fate of discharged vesicle membrane and to reveal the path by which vesicle membrane is retrieved from the plasma membrane by endocytosis. New methods for frozen tissue preparation for electron microscopy will also be developed in order to visualize the packaging of neurotransmitters into new synaptic vesicles during this vesicle membrane recycling, and to identify by immunocytochemistry the location and movements of the membrane proteins that are involved in these neurosecretory phenomena.